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Streptococcus pneumoniae Enhances Human Respiratory Syncytial Virus Infection In Vitro and In Vivo.

Nguyen DT, Louwen R, Elberse K, van Amerongen G, Yüksel S, Luijendijk A, Osterhaus AD, Duprex WP, de Swart RL - PLoS ONE (2015)

Bottom Line: Intranasal HRSV infection three days later resulted in strain-specific enhancement of HRSV replication in vivo.However, neither pneumococci nor HRSV were found to spread from the upper to the lower respiratory tract, and neither pathogen was transmitted to naive cage mates by direct contact.These results demonstrate that pneumococcal colonization can enhance subsequent HRSV infection, and provide tools for additional mechanistic and intervention studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Viroscience, Erasmus MC, Rotterdam, The Netherlands.

ABSTRACT
Human respiratory syncytial virus (HRSV) and Streptococcus pneumoniae are important causative agents of respiratory tract infections. Both pathogens are associated with seasonal disease outbreaks in the pediatric population, and can often be detected simultaneously in infants hospitalized with bronchiolitis or pneumonia. It has been described that respiratory virus infections may predispose for bacterial superinfections, resulting in severe disease. However, studies on the influence of bacterial colonization of the upper respiratory tract on the pathogenesis of subsequent respiratory virus infections are scarce. Here, we have investigated whether pneumococcal colonization enhances subsequent HRSV infection. We used a newly generated recombinant subgroup B HRSV strain that expresses enhanced green fluorescent protein and pneumococcal isolates obtained from healthy children in disease-relevant in vitro and in vivo model systems. Three pneumococcal strains specifically enhanced in vitro HRSV infection of primary well-differentiated normal human bronchial epithelial cells grown at air-liquid interface, whereas two other strains did not. Since previous studies reported that bacterial neuraminidase enhanced HRSV infection in vitro, we measured pneumococcal neuraminidase activity in these cultures but found no correlation with the observed infection enhancement in our model. Subsequently, a selection of pneumococcal strains was used to induce nasal colonization of cotton rats, the best available small animal model for HRSV. Intranasal HRSV infection three days later resulted in strain-specific enhancement of HRSV replication in vivo. One S. pneumoniae strain enhanced HRSV both in vitro and in vivo, and was also associated with enhanced syncytium formation in vivo. However, neither pneumococci nor HRSV were found to spread from the upper to the lower respiratory tract, and neither pathogen was transmitted to naive cage mates by direct contact. These results demonstrate that pneumococcal colonization can enhance subsequent HRSV infection, and provide tools for additional mechanistic and intervention studies.

No MeSH data available.


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Bacterial and virus titers of HRSV infected S. pneumoniae colonized cotton rats.(A) Experimental design. Pneumococcal carriage was induced by i.n. inoculation of cotton rats with 5x105 CFU S. pneumoniae in 10 μl at day 0. Three days later animals were infected with 1x104 TCID50 rHRSVB05EGFP(5) in PBS (10 μl). At day 8 animals were euthanized. (B) Bacterial titers eight days after induction of pneumococcal carriage. The geometric mean titer was about 2x105 CFU/ml for the different strains. Data symbols represent individual animals, bars represent geometric mean titers (GMT) per group. * p ≤ 0.05; 2-tailed Mann-Whitney U test. (C) Virus titers five days after HRSV infection. Significantly higher virus load were detected in groups with nasal carriage of S. pneumoniae strain 19F and 23F compared to mock-treated. * p ≤ 0.05; 2-tailed Mann-Whitney U test.
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pone.0127098.g002: Bacterial and virus titers of HRSV infected S. pneumoniae colonized cotton rats.(A) Experimental design. Pneumococcal carriage was induced by i.n. inoculation of cotton rats with 5x105 CFU S. pneumoniae in 10 μl at day 0. Three days later animals were infected with 1x104 TCID50 rHRSVB05EGFP(5) in PBS (10 μl). At day 8 animals were euthanized. (B) Bacterial titers eight days after induction of pneumococcal carriage. The geometric mean titer was about 2x105 CFU/ml for the different strains. Data symbols represent individual animals, bars represent geometric mean titers (GMT) per group. * p ≤ 0.05; 2-tailed Mann-Whitney U test. (C) Virus titers five days after HRSV infection. Significantly higher virus load were detected in groups with nasal carriage of S. pneumoniae strain 19F and 23F compared to mock-treated. * p ≤ 0.05; 2-tailed Mann-Whitney U test.

Mentions: In order to extrapolate our in vitro results a new in vivo pneumococcal colonization & HRSV infection model was developed. We established this model using four S. pneumoniae strains and the recombinant HRSV strain expressing EGFP (Fig 2A). Animals (n = 6/group) were intra-nasally inoculated with 5x105 CFU of pneumococci or with PBS as mock control. An inoculum volume of 10 μl was used to prevent primary inoculation of the lower respiratory tract (LRT) [34]. Animals were infected 3 days post-colonization with 1x104 50% tissue culture infectious doses (TCID50) of rHRSVB05EGFP(5) in PBS (10 μl). No samples were collected until euthanasia at day 8 (i.e. day 5 after HRSV infection) to prevent any mechanical/physical interference with bacterial colonization or viral infection and spread. Nasopharyngeal lavages and lung homogenates were collected post-mortem for isolation of pneumococci and HRSV. Nasal septum, conchae and agarose-inflated lungs were collected for immediate monitoring of EGFP+ cells [28, 32].


Streptococcus pneumoniae Enhances Human Respiratory Syncytial Virus Infection In Vitro and In Vivo.

Nguyen DT, Louwen R, Elberse K, van Amerongen G, Yüksel S, Luijendijk A, Osterhaus AD, Duprex WP, de Swart RL - PLoS ONE (2015)

Bacterial and virus titers of HRSV infected S. pneumoniae colonized cotton rats.(A) Experimental design. Pneumococcal carriage was induced by i.n. inoculation of cotton rats with 5x105 CFU S. pneumoniae in 10 μl at day 0. Three days later animals were infected with 1x104 TCID50 rHRSVB05EGFP(5) in PBS (10 μl). At day 8 animals were euthanized. (B) Bacterial titers eight days after induction of pneumococcal carriage. The geometric mean titer was about 2x105 CFU/ml for the different strains. Data symbols represent individual animals, bars represent geometric mean titers (GMT) per group. * p ≤ 0.05; 2-tailed Mann-Whitney U test. (C) Virus titers five days after HRSV infection. Significantly higher virus load were detected in groups with nasal carriage of S. pneumoniae strain 19F and 23F compared to mock-treated. * p ≤ 0.05; 2-tailed Mann-Whitney U test.
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Related In: Results  -  Collection

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pone.0127098.g002: Bacterial and virus titers of HRSV infected S. pneumoniae colonized cotton rats.(A) Experimental design. Pneumococcal carriage was induced by i.n. inoculation of cotton rats with 5x105 CFU S. pneumoniae in 10 μl at day 0. Three days later animals were infected with 1x104 TCID50 rHRSVB05EGFP(5) in PBS (10 μl). At day 8 animals were euthanized. (B) Bacterial titers eight days after induction of pneumococcal carriage. The geometric mean titer was about 2x105 CFU/ml for the different strains. Data symbols represent individual animals, bars represent geometric mean titers (GMT) per group. * p ≤ 0.05; 2-tailed Mann-Whitney U test. (C) Virus titers five days after HRSV infection. Significantly higher virus load were detected in groups with nasal carriage of S. pneumoniae strain 19F and 23F compared to mock-treated. * p ≤ 0.05; 2-tailed Mann-Whitney U test.
Mentions: In order to extrapolate our in vitro results a new in vivo pneumococcal colonization & HRSV infection model was developed. We established this model using four S. pneumoniae strains and the recombinant HRSV strain expressing EGFP (Fig 2A). Animals (n = 6/group) were intra-nasally inoculated with 5x105 CFU of pneumococci or with PBS as mock control. An inoculum volume of 10 μl was used to prevent primary inoculation of the lower respiratory tract (LRT) [34]. Animals were infected 3 days post-colonization with 1x104 50% tissue culture infectious doses (TCID50) of rHRSVB05EGFP(5) in PBS (10 μl). No samples were collected until euthanasia at day 8 (i.e. day 5 after HRSV infection) to prevent any mechanical/physical interference with bacterial colonization or viral infection and spread. Nasopharyngeal lavages and lung homogenates were collected post-mortem for isolation of pneumococci and HRSV. Nasal septum, conchae and agarose-inflated lungs were collected for immediate monitoring of EGFP+ cells [28, 32].

Bottom Line: Intranasal HRSV infection three days later resulted in strain-specific enhancement of HRSV replication in vivo.However, neither pneumococci nor HRSV were found to spread from the upper to the lower respiratory tract, and neither pathogen was transmitted to naive cage mates by direct contact.These results demonstrate that pneumococcal colonization can enhance subsequent HRSV infection, and provide tools for additional mechanistic and intervention studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Viroscience, Erasmus MC, Rotterdam, The Netherlands.

ABSTRACT
Human respiratory syncytial virus (HRSV) and Streptococcus pneumoniae are important causative agents of respiratory tract infections. Both pathogens are associated with seasonal disease outbreaks in the pediatric population, and can often be detected simultaneously in infants hospitalized with bronchiolitis or pneumonia. It has been described that respiratory virus infections may predispose for bacterial superinfections, resulting in severe disease. However, studies on the influence of bacterial colonization of the upper respiratory tract on the pathogenesis of subsequent respiratory virus infections are scarce. Here, we have investigated whether pneumococcal colonization enhances subsequent HRSV infection. We used a newly generated recombinant subgroup B HRSV strain that expresses enhanced green fluorescent protein and pneumococcal isolates obtained from healthy children in disease-relevant in vitro and in vivo model systems. Three pneumococcal strains specifically enhanced in vitro HRSV infection of primary well-differentiated normal human bronchial epithelial cells grown at air-liquid interface, whereas two other strains did not. Since previous studies reported that bacterial neuraminidase enhanced HRSV infection in vitro, we measured pneumococcal neuraminidase activity in these cultures but found no correlation with the observed infection enhancement in our model. Subsequently, a selection of pneumococcal strains was used to induce nasal colonization of cotton rats, the best available small animal model for HRSV. Intranasal HRSV infection three days later resulted in strain-specific enhancement of HRSV replication in vivo. One S. pneumoniae strain enhanced HRSV both in vitro and in vivo, and was also associated with enhanced syncytium formation in vivo. However, neither pneumococci nor HRSV were found to spread from the upper to the lower respiratory tract, and neither pathogen was transmitted to naive cage mates by direct contact. These results demonstrate that pneumococcal colonization can enhance subsequent HRSV infection, and provide tools for additional mechanistic and intervention studies.

No MeSH data available.


Related in: MedlinePlus